Phosphorylation Of MYLK/MLC2 By SIK2 Promotes Ovarian Cancer Cell Motility And Metastasis

Ovarian cancer is one of the common malignant tumors of the female reproductive system.Its incidence is lower than that of cervical cancer and endometrial cancer.However,most patients have experienced extensive metastasis of pelvic and abdominal organs at the time of discovery,so its mortality rate has always been ranked first in gynecology.The mechanism of ovarian cancer metastasis is still not fully understood.To clarify the specific mechanism and potential therapeutic targets of ovarian cancer metastasis to the abdominal cavity is an urgent problem in the field of gynecological tumors.Part one Exploration the functional study of SIK2 in promoting ovarian cell motion and metastasisObjective:To explore the expression of SIK2 in ovarian cancer tissues and effect of the migration and invasion ability of targeting SIK2 in ovarian cancer cells.We also observed the effect of cytoskeleton F-actin and cell motor protein non-muscle myosin II regulated the phosphorylation level of light chain MLC2 and cell morphology after targeting SIK2.Methods:The expression of SIK2 in ovarian cancer tissue microarrays and seven ovarian cancer cell lines was detected by immunohistochemistry and Western Blot.SIK2 was knocked down or overexpressed by siRNA or overexpression plasmids.The wound healing assay and Transwell assay were used to detect SIK2 migration and invasion ability of SKOv3 and OVCAR8 cell after regulating SIK2.Cell phalloidin staining and immunofluorescence were used to detect changes in cytoskeleton F-actin and cell motion protein MLC2-pS19 and cell morphology.Results:1.SIK2 was highly expressed in 40.9%of epithelial ovarian cancers by ovarian cancer tissue chip immunohistochemistry,and expressed in seven ovarian cancer cell lines;2.Knockdown of SIK2 inhibits wound healing and Transwell migration and invasion ability of ovarian cancer cells,while overexpression of SIK2 promotes wound healing and Transwell migration and invasion ability of ovarian cancer.3.Knockdown of SIK2 reduces the expression of MLC2-pS19,and overexpression of SIK2 increases the expression of MLC2-pS19;4.Phalloidin staining shows that knockdown of SIK2 decreased cytoskeleton F-actin expression and over-expressing SIK2 increased cytoskeleton F-actin expression;5.Knockdown and over-expressing SIK2 stable transfected cell lines found knockdown of SIK2 increased the area of cells,while overexpression of SIK2 decreased the area of cells.Conclusion:SIK2 is highly expressed in ovarian cancer tissues.SIK2 can promote the motility and metastasis ability of ovarian cancer cells.Part two SIK2 phosphorylates MYLK at Ser343 siteObjective:To explore the phosphorylation sites of MYLK directly phosphorylated by SIK2,and construct MYLK-pS343 antibody,and explore the correlation between SIK2 expression and MYLK-pS343 expression.Methods:In vitro kinase assay,mass spectrometry,immunofluorescence colocalization experiment,Co-immunoprecipitation experiment were used to detect the phosphorylation sites of MYLK by SIK2,and at the same time prepared MYLK-pS343 specific antibody,immunospotting experiment was used to detect the specificity of the antibody,Western Blot detects the expression and correlation between SIK2 and MYLK-pS343,MLC2-pS19 in seven ovarian cancer cell lines,and analysis of the correlation between SIK2 expression and MYLK-pS343 expression in ovarian cancer tissue microarrays and the survival of patients prognosis.Results:1.SIK2 cannot directly phosphorylate MLC2 and SIK2 does not affect ROCK protein expression;2.SIK2 can directly phosphorylate MYLK at Ser343 site;3.In ovarian cancer cells,SIK2 expression was positively correlated with MYLK-pS343 and MLC2-pS19;4.In ovarian cancer tissue chips,the expression of SIK2 was positively correlated with the expression of MYLK-pS343,and the prognosis of patients with high expression of SIK2 and MYLK-pS343 was poor.Conclusion:SIK2 directly phosphorylates MYLK at Ser343 sitePart three MYLK promotes ovarian cancer cell motion and metastasisObjective:To explore whether SIK2 through MYLK exerting effects on the migration and invasion ability of ovarian cancer cells and the cell motor protein non-muscle myosin II regulated the phosphorylation level of light chain MLC2.To study the effect of MYLK overexpression plasmid on MLC2 phosphorylation in SIK2 knockdown cell lines.To explore whether SIK2 has the effect of MYLK on the migration and invasion of ovarian cancer cells and the effect of cell motor protein non-muscle myosin II on the phosphorylation level of light chain MLC2,and the effect of MYLK overexpression plasmid on MLC2 phosphorylation.Methods:Transwell migration and invasion experiments were used to detect the migration and invasion ability of SKOv3 and OVCAR8 cells by loss function of MYLK,and Western Blot was used to detect the changes of cytoskeleton protein F-actin and cell motor protein MLC2-pS 19.OVCAR8-shSIK2 cells were transfected with the wild-type MYLK plasmid WT,the MYLK dephosphorylation mutant plasmid MYLK-S343A and the MYLK pseudo-phosphorylated mutant plasmid MYLK-S343E,and observe the MLC2 phosphorylation level in the SIK2 knockdown cell line.Results:1.Knockdown of MYLK inhibited the migration and invasion of ovarian cancer cells;2.Targeted siRNA knockdown of MYLK or MYLK inhibitor ML-9 reduced the expression of F-actin and MLC2-pS19;3.MYLK wild-type plasmid WT and MYLK pseudo-phosphorylation mutant plasmid MYLK-S343E can reverse the decrease of MLC2-pS19 caused by SIK2 knockdown,but MYLK dephosphorylation mutant plasmid MYLK-S343A cannot.Conclusion:SIK2 promotes the motion and metastasis of ovarian cancer cells by phosphorylating MYLK at Ser343.Part four Omental adipocytes promote ovarian cancer metastasis via phosphorylation SIK2/MYLK/MLC2 pathwayObjective:To investigate the influence of omental adipocytes on the metastasis ability of ovarian cancer cells and the influence on the SIK2/MYLK/MLC2 pathway.Methods:The omental adipocytes were isolated and cultured,and co-cultured with ovarian cancer cells SKOv3 and OVCAR8.Transwell experiment was used to observe the migration and invasion ability of co-cultured SKOv3 and OVCAR8 cells.Western Blot was used to detect SIK2/MYLK/MLC2 phosphorylation levels in co-cultured ovarian cancer cells.Results:1.Omental adipocytes promoted the migration and invasion of ovarian cancer cells;2.Omental adipocytes promoted the phosphorylation of SIK2/MYLK/MLC2.Conclusion:Omental adipocytes promote the metastasis of ovarian cancer cells through the phosphorylation SIK2/MYLK/MLC2 pathwayPart Five SIK2 promotes the motion and metastasis of ovarian cancer cells via phosphorylation MYLK/MLC2 pathway in vivoObjective:To explore the effect of SIK2 on ovarian cancer metastasis and the level of MYLK/MLC2 phosphorylation in animal experiments.Methods:The SCID mouse orthotopic ovarian cancer xenograft model was constructed with luciferase-carrying stable OVCAR8-shNC-luc cells and OVCAR8-shSIK2-luc cells,and animal live imaging was used to observe the effect of SIK2 knockdown on the primary ovarian cancer and metastases,immunohistochemistry and Western Blot were used to detect the changes in the phosphorylation levels of MYLK and MLC2 in animal tumor tissues by SIK2 knockdown.Results:1.The fluorescence intensity and metastatic area of nude mice in the OVCAR8-shSIK2-Luc cell group injected in situ on the ovary were significantly smaller than those in the OVCAR8-shNC-Luc cell group injected in situ;and the weights of primary and metastatic foci after SIK2 knockdown were significantly reduced;2.After SIK2 knockdown,the expression of MYLK-pS343 and MLC2-pS19 in animal tumor tissues was significantly reduced.Conclusion: SIK2 promotes the motion and metastasis of ovarian cancer cells via phosphorylation MYLK/MLC2 pathway in vivo.